细胞上清外泌体提取试剂盒(沉淀法)

碧云天生产的细胞上清外泌体提取试剂盒(沉淀法) (Exosome Isolation Kit from Cell Culture Media by Precipitation Method)，也称为细胞培养液上清外泌体提取试剂盒、外泌体纯化试剂盒、外泌体抽提试剂盒、Total Exosome Isolation Kit，是一种高效、便捷的用于细胞培养液上清中外泌体或其它细胞外囊泡提取的试剂盒，仅需简单的较低速度离心即可从样品中分离出大量完整的、纯度较高的外泌体。使用本试剂盒提取的外泌体可用于蛋白分析、核酸分析、Western blot、PCR、RNA提取、高通量测序、外泌体粒径和浓度分析即纳米颗粒示踪分析(Nanoparticle tracking analysis, NTA)、电镜分析、细胞共培养等实验。

外泌体(Exosome)是膜包裹的细胞外囊泡(Extracellular vesicles, EVs)，直径约为40-160nm，具有脂质双分子层结构，天然存在于血液、尿液、脑脊液，以及体外培养细胞的上清液中[1]，几乎所有类型的细胞都可以产生并释放外泌体[2]。如图1所示，细胞膜内吞(Endocytosis)依次形成初级内体(Early sorting endosome, ESE)、次级内体(Late sorting endosome, LSE)和多囊泡体(Multivesicular body, MVB)，其中多囊泡体包含腔内囊泡(Intraluminal vesicles, ILVs)。多囊泡体与细胞膜融合形成外泌体，外泌体携带多种来自其母体细胞的成分(包括核酸、蛋白质、脂类、糖类和代谢物等)释放到胞外基质中[3]。外泌体可以被附近或远距离的细胞识别和融合，是细胞间进行相互调控的重要媒介，参与了癌症、神经退行性病变和炎症性疾病等多种疾病的发病过程，影响细胞多方面的功能[4-5]。

图1.外泌体的形成原理及其携带的母体细胞成分示意图。

常规的细胞外囊泡提取方法有超速离心法(Ultracentrifugation/UC)、沉淀法(Precipitation)、尺寸排阻色谱法(Size-exclusion chromatography/SEC)、超滤法(Ultrafiltration/UF)、免疫亲和分离法(Immunoaffinity capture/IAC)等。各提取方法的原理、优缺点请参考下表[6]。

Methods	Separation principle	Advantages	Disadvantages
Ultracentrifugation (超速离心法)	Density; Floatation speed (in case of density gradient)	Most widely used as the Gold Standard; Robust method; Possibility to identify subpopulations when density gradient is applied.	Heavy workload; Might lead to ruptured EVs; Low EV yield; Results are difficult to compare between different studies due to different rotor types and centrifugation speeds; Large volume of starting material required.
Precipitation (沉淀法)	Solubility	User-friendly method; High EV yield; Many kits commercially available; Relatively cheap method; Possibility for clinical application.	High risk of co-isolating non-vesicular contaminants; Polymers may be problematic for some downstream applications; Might not be suitable for performing functional studies.
Size-exclusion chromatography (尺寸排阻色谱法)	Hydrodynamic radius	Fast and simple; EV integrity is maintained during isolation; May be used in upscaling processes.	Potentially significant contamination with non-EV particles (e.g. lipoproteins、albumin).
Ultrafiltration (超滤法)	Hydrodynamic radius	Can be very well combined with other methods; Easy to perform in every lab.	Filter type and ultrafiltration method influence EV yield and composition considerably; Rather limited resolution.
Field-flow fractionation (场流分离法)	Hydrodynamic radius; Electrophoretic mobility	Can identify smaller EV subsets (e.g. exomeres).	Specific equipment required and the associated expertise; Cannot be used as a stand-alone method.
Immunoaffinity capture (免疫亲和分离法)	Surface markers	Very pure EV isolations; Can be combined with light scattering flow cytometry; Capable of isolating subpopulations.	Currently not suitable for a complete isolation of all EVs; Expensive.
Microfluidics (微流控技术)	Surface markers; Hydrodynamic radius; Density	EV isolation can be combined with EV characterization; Subpopulations can be isolated; Small sample volumes can be used for EV isolation.	Requires a specific level of expertise; Not suitable for preparative purposes (e.g. therapeutic applications).
Membrane affinity methods (膜亲和法)	Membrane affinity	Is able to isolate all EVs instead of only EV subpopulations.	Cannot discriminate between EVs and cell(s)fragments.

本产品采用多聚物沉淀法，设备要求低、操作方便、提取时间短、样品起始量低、提取效率高、外泌体形态比较完整。

本产品无RNA酶、无DNA酶、无蛋白酶。使用本产品提取得到的外泌体可以用于RNA、DNA及蛋白的各种应用。使用本产品提取的外泌体，经Western blot检测外泌体标志性蛋白的效果参考图2。

图2.细胞上清外泌体提取试剂盒(沉淀法) (C3620)提取的外泌体经Western blot验证的效果图。无血清培养液培养的293H细胞上清，经本试剂盒提取外泌体后，通过Western blot检测外泌体标志性蛋白HSP70和TSG101。实际效果会因实验条件、检测仪器等的不同而存在差异，图中效果仅供参考。

按使用说明操作，每次提取1ml样品需190μl的细胞上清外泌体提取试剂，一个小包装的本试剂盒可使用约50次，一个中包装的本试剂盒可使用约250次。

包装清单：

产品编号	产品名称	包装
C3620S	细胞上清外泌体提取试剂	10ml
C3620M	细胞上清外泌体提取试剂	50ml
－	说明书	1份

保存条件：

4ºC保存，一年有效。室温保存，一个月有效。-20ºC保存，至少两年有效。

注意事项：

本产品较为粘稠，使用时需完全混合均匀后再吸取，并保证吸取体积准确。

本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。

为了您的安全和健康，请穿实验服并戴一次性手套操作。